Our studies concentrate on proteins that traffick in the late endocytic pathway in compartments that effect cellular cholesterol and glycolipid mobilization (NPC1, NPC2, MLN64, ML4, Apo D, and CD63). Apolipoprotein D: role in cellular cholesterol trafficking. We showed that sterol accumulation in lysosomes is linked to a parallel enrichment of this organelle with apoD suggesting that apoD mediates a cellular function in the trafficking of LDL derived cholesterol through lysosomes. Apo D colocalizes with cholesterol in Niemann Pick C-/-cells and WT cells treated with LDL and progesterone. We investigated the interaction of NPC1 protein with apoD using photobleaching fluorescence energy transfer (pbFRET) and found that apoD interacts with NPC1 only under conditions of cholesterol loading. The interaction of apoD with NP-C1 may represent a modulation in NP-C1 trafficking of endocytosed cholesterol. Apolipoprotein D (-/-) mouse fibroblasts: Faulty regulation of low density lipoprotein uptake and metabolism. Cultured fibroblasts from apolipoprotein D (-/-) mice have abnormal metabolism of low-density lipoprotein. In response to uptake of human low density lipoprotein (LDL) there was an abnormal accumulation of neutral lipid and unesterified cholesterol in lysosomes of apoD (-/-) mouse embryonic fibroblasts when compared with normal embryonic fibroblasts. With con-focal fluorescence microscopy, neutral lipid was visualized with nile red, lysosomes were visualized with antibodies against lysosomal associated membrane protein (LAMP) and unesterified cholesterol was visualized in lysosomes with filipin fluorescence. Biochemical analysis of triacylglycerol synthesis in normal and apoD (-/-) fibroblasts showed no stimulation by LDL uptake nor differences in accumulation between normal and mutant fibroblasts. However, biochemical analysis did show that apoD (-/-) fibroblasts synthesized more cholesterol ester than normal fibroblasts, during 4h and 24h incubations with LDL, when neutral lipid and unesterified cholesterol accumulated in lysosomes of these cells. ApoD (-/-) fibroblasts were incubated with LDL in the presence of CI-976, an inhibitor of acyl-coenzymeA: cholesterol acyl transferase (ACAT), the enzyme necessary for cholesterol esterification in the endoplasmic reticulum. Mutant cells incubated with LDL during ACAT inhibition did not form the biochemically measured pool of cholesterol ester whereas, the cytochemically visualized pool of neutral lipid in lysosomes did accumulate. Thus, the ACAT inhibitor sensitive pool of cholesterol ester is present in the ER and the nile red positive droplets in lysosomes are a second and separate pool of cholesterol ester formed in mutant cells by uptake of LDL. This CE pool in lysosomes of mutant cells could be unhydrolyzed LDL or lysosomal re-esterification of cholesterol derived from LDL. The presence of unesterified cholesterol in lysosomes seen by filipin fluorescence suggested that lysosomal hydrolysis of LDL could occur in mutant cells lacking endogeneous Apo D. However, since human LDL contains some human apolipoprotein D, we incubated apo D (-/-) mutant mouse cells with human LDL that had been pre- incubated ("neutralized") with antibodies to human apolipoprotein D. Mutant mouse fibroblasts incubated with "apo D neutralized" human LDL showed accumulation of neutral lipid but not unesterified cholesterol. Thus, we suggest that the unesterified cholesterol seen in lysosomes of apoD (-/-) fibroblasts results from hydrolysis of LDL by the activity of lysosomal acid lipase (LAL) in concert with exogeneously derived LDL-human ApoD. We are now investigating, in apo D(-/-) cells, the hydrolytic activity of LAL and the intracellular trafficking of cholesterol derived from artificial cholesterol-ester emulsions to further explore the role of Apo D in lysosomal hydrolysis of low density lipoproteins. Effect of ApoD on net cholesterol balance in the mouse: Our studies on normal and apo D (-/-) mice given a hypercholesterylemic diet show that the absence of apoD protects excessive cholesterol build up in the mutant mice. We are assaying livers from the normal and apoD mutant mice with biochemistry and cytochemistry to show increased accumulation of cholesterol. CD63 (LIMP 1): CD63 resides in the endocytic pathway and is an integral protein of cellular multivesicular bodies organelles that supply membrane flow through the late endocytic pathway by forming tubular extensions that communicate with lysosomes. We are currently investigating the role of CD63 in the formation of multivesicular bodies. Our initial approach is to label early endosomes with exogeneously derived tracer molecules at low temperature and then shift to a higher temperature to label late endosomes and lysosomes. The effect of knockdown of CD63 via siRNA on formation of multivesicular bodies will be quantitated with electron microscopy. These studies question whether multivesicular bodies are critical to membrane flow in the late endosomal tubular pathway that traffick exogeneously derived cholesterol and glycolipids to lysosomes and from the plasma membrane